Biomedical Insights that Inform the Diagnosis of ME/CFS Printed Edition of the Special Issue Published in Diagnostics www.mdpi.com/journal/diagnostics Brett A. Lidbury and Paul R. Fisher Edited by Biomedical Insights that Inform the Diagnosis of ME/CFS Biomedical Insights that Inform the Diagnosis of ME/CFS Special Issue Editors Brett A. Lidbury Paul R. Fisher MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Special Issue Editors Brett A. Lidbury The Australian National University Australia Paul R. Fisher La Trobe University Australia Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Diagnostics (ISSN 2075-4418) (available at: https://www.mdpi.com/journal/diagnostics/special issues/ME CFS). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Article Number , Page Range. ISBN 978-3-03928-390-3 (Pbk) ISBN 978-3-03928-391-0 (PDF) c © 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Special Issue Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Brett A. Lidbury and Paul R. Fisher Biomedical Insights that Inform the Diagnosis of ME/CFS Reprinted from: Diagnostics 2020 , 10 , 92, doi:10.3390/diagnostics10020092 . . . . . . . . . . . . . 1 Mateo Cortes Rivera, Claudio Mastronardi, Claudia T. Silva-Aldana, Mauricio Arcos-Burgos and Brett A. Lidbury Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Comprehensive Review Reprinted from: Diagnostics 2019 , 9 , 91, doi:10.3390/diagnostics9030091 . . . . . . . . . . . . . . . 5 Daniel Missailidis, Sarah J. Annesley and Paul R. Fisher Pathological Mechanisms Underlying Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Reprinted from: Diagnostics 2019 , 9 , 80, doi:10.3390/diagnostics9030080 . . . . . . . . . . . . . . . 39 Eiren Sweetman, Alex Noble, Christina Edgar, Angus Mackay, Amber Helliwell, Rosamund Vallings, Margaret Ryan and Warren Tate Current Research Provides Insight into the Biological Basis and Diagnostic Potential for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) Reprinted from: Diagnostics 2019 , 9 , 73, doi:10.3390/diagnostics9030073 . . . . . . . . . . . . . . . 59 Frank Twisk Myalgic Encephalomyelitis or What? The International Consensus Criteria Reprinted from: Diagnostics 2019 , 9 , 1, doi:10.3390/diagnostics9010001 . . . . . . . . . . . . . . . 73 Mark Vink and Friso Vink-Niese Work Rehabilitation and Medical Retirement for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients. A Review and Appraisal of Diagnostic Strategies Reprinted from: Diagnostics 2019 , 9 , 124, doi:10.3390/diagnostics9040124 . . . . . . . . . . . . . . 81 Carly S. Holtzman, Shaun Bhatia, Joseph Cotler and Leonard A. Jason Assessment of Post-Exertional Malaise (PEM) in Patients with Myalgic Encephalomyelitis (ME) and Chronic Fatigue Syndrome (CFS): A Patient-Driven Survey Reprinted from: Diagnostics 2019 , 9 , 26, doi:10.3390/diagnostics9010026 . . . . . . . . . . . . . . . 115 Luis Nacul, Barbara de Barros, Caroline C. Kingdon, Jacqueline M. Cliff, Taane G. Clark, Kathleen Mudie, Hazel M. Dockrell and Eliana M. Lacerda Evidence of Clinical Pathology Abnormalities in People with Myalgic Esncephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) from an Analytic Cross-Sectional Study Reprinted from: Diagnostics 2019 , 9 , 41, doi:10.3390/diagnostics9020041 . . . . . . . . . . . . . . . 129 Brett A. Lidbury, Badia Kita, Alice M. Richardson, Donald P. Lewis, Edwina Privitera, Susan Hayward, David de Kretser and Mark Hedger Rethinking ME/CFS Diagnostic Reference Intervals via Machine Learning, and the Utility of Activin B for Defining Symptom Severity Reprinted from: Diagnostics 2019 , 9 , 79, doi:10.3390/diagnostics9030079 . . . . . . . . . . . . . . . 145 Neil R. McGregor, Christopher W. Armstrong, Donald P. Lewis and Paul R. Gooley Post-Exertional Malaise Is Associated with Hypermetabolism, Hypoacetylation and Purine Metabolism Deregulation in ME/CFS Cases Reprinted from: Diagnostics 2019 , 9 , 70, doi:10.3390/diagnostics9030070 . . . . . . . . . . . . . . . 167 v Alex A. Kashi, Ronald W. Davis and Robert D. Phair The IDO Metabolic Trap Hypothesis for the Etiology of ME/CFS Reprinted from: Diagnostics 2019 , 9 , 82, doi:10.3390/diagnostics9030082 . . . . . . . . . . . . . . . 179 vi About the Special Issue Editors Brett A. Lidbury , Associate Professor, B.S. (Hons), Ph.D., FFSc (RCPA) Brett Lidbury is an Associate Professor with the National Centre for Epidemiology and Population Health (NCEPH), Research School of Population Health, The Australian National University (ANU) Canberra, Australia. He brings a laboratory background to his current role, with previous research programmes in infectious disease focused on virus–host interaction and viral pathogenesis. Discoveries in these fields included the elucidation of mechanisms for inflammatory regulation by Ross River virus (RRV, a mosquito-transmitted alphavirus), macrophage persistence by RRV, and muscle-joint pathology. RRV is associated with post-viral fatigue in Australia, and results from RRV research led to the current investigations into ME/CFS aetiology, which has taken a translational route with machine learning on patient data, as well as volunteer recruitment that requires clinical collaboration. Contributions to ME/CFS research include the identification of activin B as a potential serum biomarker, both alone and in combination with pathology test markers. The application of machine learning to pathology results and clinical observations is an ongoing research interest, with the aim of redefining diagnostic reference intervals that support an ME/CFS laboratory diagnosis and therefore assist clinicians.Lidbury is a member of the ME/CFS Discovery Research Network (MDRN) and is an investigator on the J.J. Mason Foundation-funded ME/CFS Biobank-Database programme in Australia. Paul R. Fisher , Professor, BSc(Hons)(Qld), MSc(Qld), PhD(ANU) Professor Fisher has been the Professor and Head of Microbiology at La Trobe University, Melbourne, Australia since 2004. He has studied neurodegenerative and neurological disease, mitochondrial biology, and the roles of mitochondria in disease using two model systems to understand the cytopathologic pathways involved: 1. The eukaryotic microbe Dictyostelium , which is one of 10 model organisms recognized by the NIH for their value in biomedical research. Dictyostelium provides a tractable molecular genetic model for mitochondrial disease and neurodegenerative diseases; he is a world leader in this research. Using the Dictyostelium model, he specifically manipulates (separately and together) known disease genes and genes encoding proteins hypothesized to be involved in the associated cytopathological pathways. He then uses biochemical, molecular, and cell biological assays to determine the cellular consequences of disturbances in these pathways. The results support pathological roles for cellular stress signalling pathways in mitochondrial and neurodegenerative diseases. 2. Cultured cell lines from human disease patients and healthy control individuals. His laboratory’s growing collection of cell lines currently includes a total of ca. 300 different lymphoblast (immortalized B cells) and fibroblast cell lines from healthy individuals and patients with Parkinson’s disease, fragile-X-associated tremor and ataxia syndrome (FXTAS), and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The aim is to study the roles of mitochondria and cellular stress signalling pathways in these diseases. This research requires advanced expertise in mitochondrial and molecular biology, biochemistry, and cell physiology, including the complete array of modern molecular techniques, as well as in the diverse statistical methods in the data analysis. vii ȱ ȱ Diagnostics ȱ 2020 , ȱ 10 , ȱ 92; ȱ doi:10.3390/diagnostics10020092 ȱ www.mdpi.com/journal/diagnostics ȱ Editorial ȱ Biomedical ȱ Insights ȱ that ȱ Inform ȱ the ȱ Diagnosis ȱ of ȱ ME/CFS ȱ Brett ȱ A. ȱ Lidbury ȱ 1, * ȱ and ȱ Paul ȱ R. ȱ Fisher ȱ 2 ȱ 1 ȱ National ȱ Centre ȱ for ȱ Epidemiology ȱ and ȱ Population ȱ Health, ȱ Research ȱ School ȱ of ȱ Population ȱ Health, ȱ Australian ȱ National ȱ University, ȱ Canberra, ȱ ACT ȱ 2601, ȱ Australia ȱ 2 ȱ Department ȱ of ȱ Physiology, ȱ Anatomy, ȱ and ȱ Microbiology, ȱ La ȱ Trobe ȱ University, ȱ Melbourne, ȱ VIC ȱ 3086, ȱ Australia; ȱ P.Fisher@latrobe.edu.au ȱ * ȱ Correspondence: ȱ brett.lidbury@anu.edu.au Received: ȱ 6 ȱ February ȱ 2020; ȱ Accepted: ȱ 6 ȱ February ȱ 2020; ȱ Published: ȱ 8 ȱ February ȱ 2020 ȱ ȱ It ȱ is ȱ well ȱ known ȱ that ȱ myalgic ȱ encephalomyelitis ȱ (ME) ȱ and ȱ chronic ȱ fatigue ȱ syndrome ȱ (CFS), ȱ whether ȱ considered ȱ as ȱ separate ȱ diseases ȱ or ȱ as ȱ the ȱ one ȱ chronic ȱ syndrome, ȱ continue ȱ to ȱ generate ȱ debate. ȱ Discussions ȱ on ȱ language, ȱ definitions ȱ and ȱ theoretical ȱ parameters ȱ continue, ȱ but ȱ whatever ȱ your ȱ position, ȱ one ȱ can ȱ now ȱ agree ȱ that ȱ ME ȱ and/or ȱ CFS ȱ (referred ȱ to ȱ hereafter ȱ as ȱ ME/CFS) ȱ is ȱ a ȱ disease ȱ with ȱ a ȱ physiological ȱ basis, ȱ rooted ȱ in ȱ biochemical ȱ and ȱ molecular ȱ dysfunction ȱ in ȱ the ȱ cells ȱ of ȱ sick ȱ individuals, ȱ and ȱ not ȱ attitudes ȱ that ȱ can ȱ be ȱ alleviated ȱ by ȱ psychological ȱ therapies. ȱ As ȱ a ȱ result, ȱ biomedical ȱ imperatives ȱ must ȱ now ȱ become ȱ the ȱ focus ȱ of ȱ research ȱ enquiry ȱ in ȱ order ȱ to ȱ find ȱ clinically ȱ translatable ȱ answers ȱ as ȱ soon ȱ as ȱ possible. ȱ This ȱ book ȱ is ȱ intended ȱ as ȱ a ȱ landmark ȱ volume ȱ to ȱ mark ȱ this ȱ shift ȱ in ȱ thinking ȱ and ȱ to ȱ consolidate ȱ recent ȱ fundamental ȱ discoveries ȱ and ȱ biomedical ȱ insights ȱ as ȱ pathways ȱ towards ȱ tangible ȱ diagnostics, ȱ and ȱ eventual ȱ ME/CFS ȱ treatments. ȱ Australian ȱ researchers, ȱ with ȱ their ȱ collaborators ȱ locally ȱ and ȱ abroad, ȱ have ȱ been ȱ at ȱ the ȱ forefront ȱ of ȱ discovery ȱ in ȱ the ȱ biomedical ȱ realm, ȱ and ȱ this ȱ book ȱ draws ȱ together ȱ fundamental ȱ and ȱ applied ȱ insights ȱ that ȱ have ȱ emerged ȱ from ȱ scientific ȱ and ȱ clinical ȱ enquiry. ȱ The ȱ consolidation ȱ of ȱ up Ȭ to Ȭ date ȱ insights ȱ into ȱ ME/CFS ȱ was ȱ catalysed ȱ by ȱ a ȱ conference ȱ (https://www.emerge. ȱ org.au/symposium#.XbIv2iVS Ȭ 3c) ȱ in ȱ March ȱ 2019 ȱ (Geelong, ȱ Australia), ȱ hosted ȱ by ȱ Emerge ȱ Australia, ȱ and ȱ several ȱ chapters ȱ in ȱ this ȱ volume ȱ are ȱ based ȱ on ȱ presentations ȱ from ȱ this ȱ meeting. ȱ Section ȱ I—Reviews, ȱ Commentaries ȱ and ȱ Opinions ȱ The ȱ book ȱ is ȱ arranged ȱ into ȱ two ȱ sections, ȱ with ȱ the ȱ first ȱ presenting ȱ the ȱ up Ȭ to Ȭ date ȱ commentaries ȱ and ȱ reviews ȱ of ȱ the ȱ broader ȱ ME/CFS ȱ literature. ȱ Cortes ȱ Rivera ȱ et ȱ al. ȱ [1] ȱ provide ȱ a ȱ “comprehensive ȱ review” ȱ of ȱ the ȱ field ȱ that ȱ takes ȱ us ȱ from ȱ some ȱ of ȱ the ȱ early ȱ recorded ȱ outbreaks, ȱ attempts ȱ to ȱ recognise ȱ the ȱ aetiology ȱ of ȱ disease, ȱ a ȱ summary ȱ of ȱ ME/CFS ȱ clinical ȱ and ȱ population ȱ features, ȱ and ȱ impacts ȱ on ȱ contemporary ȱ biomedical ȱ thinking. ȱ The ȱ expertise ȱ of ȱ some ȱ authors ȱ was ȱ reflected ȱ also ȱ by ȱ valuable ȱ commentary ȱ on ȱ the ȱ genetic ȱ basis ȱ of ȱ ME/CFS, ȱ including ȱ epigenetic ȱ studies, ȱ with ȱ interesting ȱ lessons ȱ for ȱ complex ȱ disease ȱ in ȱ general. ȱ Central ȱ to ȱ the ȱ book’s ȱ theme ȱ on ȱ “Biomedical ȱ Insights ȱ ....” ȱ are ȱ discoveries ȱ concerning ȱ ME/CFS ȱ patho Ȭ mechanism. ȱ Missailidis ȱ et ȱ al. ȱ [2] ȱ provides ȱ the ȱ required ȱ update ȱ on ȱ ME/CFS ȱ pathology ȱ as ȱ explained ȱ at ȱ the ȱ cellular ȱ level ȱ via ȱ biochemical ȱ and ȱ molecular ȱ alterations ȱ to ȱ function. ȱ Considering ȱ the ȱ array ȱ of ȱ triggers/causes ȱ of ȱ ME/CFS, ȱ but ȱ a ȱ similar ȱ range ȱ of ȱ symptoms, ȱ they ȱ suggest ȱ that ȱ patient ȱ stratification ȱ along ȱ these ȱ definitions ȱ is ȱ essential ȱ to ȱ understanding ȱ the ȱ molecular ȱ links ȱ to ȱ the ȱ whole ȱ person’s ȱ clinical ȱ picture. ȱ The ȱ commentary ȱ by ȱ Sweetman ȱ et ȱ al. ȱ [3] ȱ nicely ȱ follows ȱ the ȱ general ȱ reviews, ȱ and ȱ squarely ȱ addresses ȱ the ȱ question ȱ of ȱ whether ȱ biomedical ȱ science ȱ has ȱ assisted ȱ the ȱ clinic, ȱ with ȱ their ȱ views ȱ on ȱ the ȱ biomedical ȱ potential ȱ of ȱ fundamental ȱ discoveries ȱ augmented ȱ by ȱ the ȱ presentation ȱ of ȱ data ȱ from ȱ their ȱ own ȱ research ȱ laboratory. ȱ The ȱ answer, ȱ not ȱ surprisingly, ȱ is ȱ yes ȱ and ȱ no. ȱ However, ȱ they ȱ venture ȱ opinions ȱ on ȱ the ȱ frustrations ȱ experienced ȱ by ȱ both ȱ patients ȱ and ȱ clinicians ȱ due ȱ to ȱ “ ... ȱ a ȱ long ȱ period ȱ of ȱ 1 Diagnostics ȱ 2020 , ȱ 10 , ȱ 92 ȱ ȱ debate ȱ among ȱ the ȱ health ȱ profession ȱ about ȱ the ȱ true ȱ nature ȱ of ȱ the ȱ illness. ȱ It ȱ has ȱ hindered ȱ funding ȱ for ȱ much ȱ needed ȱ research ȱ and ȱ has ȱ created ȱ inertia ȱ for ȱ researchers ȱ to ȱ join ȱ the ȱ research ȱ effort”, ȱ while ȱ acknowledging ȱ the ȱ valuable ȱ discoveries ȱ of ȱ recent ȱ years, ȱ and ȱ the ȱ potential ȱ for ȱ progress ȱ in ȱ the ȱ near ȱ future. ȱ Such ȱ views ȱ are ȱ important ȱ to ȱ express, ȱ as ȱ they ȱ give ȱ impetus ȱ to ȱ a ȱ focused ȱ research ȱ effort, ȱ in ȱ spite ȱ of ȱ ongoing ȱ debate. ȱ Capturing ȱ an ȱ element ȱ of ȱ the ȱ debate, ȱ Twisk ȱ helpfully ȱ highlights ȱ a ȱ range ȱ of ȱ issues ȱ when ȱ considering ȱ ME ȱ as ȱ compared ȱ to ȱ the ȱ ME Ȭ ICC ȱ definitions ȱ (2011 ȱ International ȱ Consensus ȱ Criteria) ȱ [4]. ȱ In ȱ Twisk’s ȱ view, ȱ the ȱ ICC ȱ has ȱ helpfully ȱ abandoned ȱ the ȱ “CFS” ȱ nomenclature, ȱ but ȱ continuing ȱ confusion ȱ exists ȱ since ȱ ME ȱ and ȱ ME Ȭ ICC ȱ definitions ȱ have ȱ not ȱ been ȱ harmonised. ȱ To ȱ complete ȱ the ȱ review ȱ and ȱ commentary ȱ section ȱ of ȱ the ȱ book, ȱ Vink ȱ and ȱ Vink Ȭ Niese ȱ [5] ȱ review ȱ the ȱ situation ȱ pertaining ȱ to ȱ ME/CFS ȱ impairment ȱ and ȱ disability ȱ in ȱ relation ȱ to ȱ work, ȱ with ȱ a ȱ focus ȱ on ȱ diagnostic ȱ strategies ȱ and ȱ prognosis, ȱ as ȱ measured ȱ by ȱ the ȱ style ȱ and ȱ intensity ȱ of ȱ work ȱ possible ȱ post ȱ diagnosis. ȱ Of ȱ particular ȱ value ȱ is ȱ that ȱ the ȱ article ȱ draws ȱ upon ȱ the ȱ experience ȱ of ȱ one ȱ author ȱ (Vink), ȱ who ȱ is ȱ an ȱ occupational ȱ physician ȱ expert ȱ in ȱ the ȱ evaluation ȱ of ȱ patient ȱ disability, ȱ and ȱ discusses ȱ how ȱ to ȱ best ȱ facilitate ȱ a ȱ return ȱ to ȱ the ȱ workplace. ȱ To ȱ our ȱ knowledge, ȱ this ȱ is ȱ the ȱ first ȱ occasion ȱ that ȱ a ȱ specific ȱ examination ȱ of ȱ ME/CFS ȱ and ȱ its ȱ occupational ȱ impact ȱ has ȱ been ȱ published. ȱ Not ȱ only ȱ is ȱ the ȱ evidence ȱ reviewed, ȱ but ȱ advice ȱ is ȱ provided ȱ on ȱ how ȱ best ȱ to ȱ prepare ȱ ME/CFS ȱ patients ȱ for ȱ a ȱ return ȱ to ȱ work, ȱ if ȱ at ȱ all ȱ possible. ȱ Section ȱ II—Research ȱ Results—Biomedical ȱ Insights ȱ and ȱ Diagnostics ȱ The ȱ following ȱ section ȱ will ȱ highlight ȱ results ȱ obtained ȱ from ȱ primary ȱ biomedical ȱ research ȱ inquiry, ȱ and ȱ as ȱ such, ȱ represents ȱ contemporary ȱ thinking ȱ on ȱ the ȱ mechanisms ȱ of ȱ disease. ȱ For ȱ complex ȱ diseases ȱ like ȱ ME/CFS, ȱ research ȱ from ȱ diverse ȱ perspectives ȱ is ȱ essential. ȱ Section ȱ II ȱ starts ȱ with ȱ a ȱ qualitative ȱ study ȱ based ȱ on ȱ surveys ȱ and ȱ clinical ȱ observations, ȱ moving ȱ to ȱ new ȱ ways ȱ of ȱ using ȱ pathology ȱ laboratory ȱ data ȱ to ȱ provide ȱ marker ȱ patterns ȱ to ȱ assist ȱ diagnosis ȱ (as ȱ well ȱ as ȱ understand ȱ aetiology), ȱ with ȱ the ȱ final ȱ chapters ȱ highlighting ȱ exciting ȱ developments ȱ at ȱ the ȱ metabolomic ȱ and ȱ cellular ȱ levels ȱ of ȱ function. ȱ Holtzman ȱ et ȱ al. ȱ [6] ȱ developed ȱ a ȱ survey, ȱ with ȱ community ȱ collaboration, ȱ to ȱ specifically ȱ assess ȱ PEM ȱ (post Ȭ exertional ȱ malaise). ȱ The ȱ surveys ȱ took ȱ the ȱ form ȱ of ȱ self Ȭ report ȱ questionnaires ȱ that ȱ were ȱ subsequently ȱ completed ȱ by ȱ over ȱ 1500 ȱ members ȱ of ȱ the ȱ patient ȱ community ȱ (35 ȱ countries, ȱ 41.1% ȱ from ȱ the ȱ USA). ȱ In ȱ the ȱ opinions ȱ of ȱ consulted ȱ community ȱ members, ȱ the ȱ most ȱ valuable ȱ PEM ȱ domains ȱ included ȱ onset ȱ triggers, ȱ timing ȱ and ȱ duration, ȱ the ȱ contribution ȱ of ȱ “personal ȱ characteristics”, ȱ among ȱ other ȱ factors ȱ not ȱ previously ȱ investigated. ȱ The ȱ authors ȱ also ȱ proposed ȱ their ȱ study ȱ as ȱ a ȱ model ȱ of ȱ community ȱ collaboration, ȱ with ȱ valuable ȱ outcomes ȱ for ȱ patients, ȱ while ȱ declaring ȱ that ȱ they ȱ lacked ȱ knowledge ȱ of ȱ what ȱ case ȱ definitions ȱ were ȱ applied ȱ to ȱ individuals ȱ in ȱ the ȱ study ȱ cohort, ȱ and ȱ did ȱ not ȱ seek ȱ independent ȱ evaluation. ȱ As ȱ alluded ȱ to ȱ earlier, ȱ the ȱ variety ȱ of ȱ case ȱ definitions/criteria ȱ continues ȱ to ȱ bedevil ȱ progress ȱ on ȱ ME/CFS. ȱ For ȱ patients ȱ who ȱ fulfil ȱ the ȱ diagnostic ȱ clinical ȱ criteria ȱ for ȱ ME/CFS, ȱ a ȱ feature ȱ of ȱ laboratory ȱ (pathology) ȱ tests ȱ is ȱ that ȱ all ȱ results ȱ across ȱ blood ȱ and ȱ biochemical ȱ markers ȱ report ȱ within ȱ the ȱ analyte ȱ reference ȱ intervals, ȱ suggesting ȱ no ȱ physiological ȱ dysfunction ȱ (but ȱ remain ȱ useful ȱ for ȱ excluding ȱ other ȱ health ȱ conditions). ȱ Nacul ȱ et ȱ al. ȱ [7] ȱ have ȱ confirmed ȱ this ȱ observation ȱ in ȱ the ȱ records ȱ of ȱ UK ȱ ME/CFS ȱ Biobank ȱ (UKMEB) ȱ participants, ȱ but ȱ found ȱ that ȱ for ȱ a ȱ normally ȱ non Ȭ requested ȱ blood ȱ test ȱ marker, ȱ creatine ȱ kinase ȱ (CK), ȱ severe ȱ cases ȱ had ȱ a ȱ significantly ȱ ( p ȱ < ȱ 0.001) ȱ reduced ȱ serum ȱ concentration ȱ compared ȱ to ȱ healthy ȱ controls, ȱ with ȱ fluctuations ȱ in ȱ CK ȱ concentrations ȱ associated ȱ with ȱ symptom ȱ severity. ȱ Serum ȱ CK ȱ concentration ȱ variation ȱ persisted ȱ despite ȱ correction ȱ for ȱ disease ȱ duration, ȱ age, ȱ sex, ȱ and ȱ so ȱ on, ȱ encouraging ȱ further ȱ investigation ȱ of ȱ CK ȱ as ȱ a ȱ diagnostic ȱ marker. ȱ The ȱ absence ȱ of ȱ pathology ȱ test ȱ results ȱ outside ȱ of ȱ the ȱ reference ȱ intervals ȱ was ȱ reported ȱ also ȱ by ȱ Lidbury ȱ et ȱ al. ȱ [8] ȱ for ȱ an ȱ Australian ȱ cohort ȱ recruited ȱ from ȱ the ȱ Melbourne ȱ region. ȱ For ȱ this ȱ investigation, ȱ the ȱ machine ȱ learning ȱ (ML) ȱ algorithm ȱ random ȱ forest ȱ (RF) ȱ was ȱ applied ȱ to ȱ identify ȱ predictor ȱ patterns ȱ from ȱ the ȱ pathology ȱ results, ȱ both ȱ for ȱ the ȱ direct ȱ comparison ȱ of ȱ ME/CFS ȱ to ȱ healthy ȱ control ȱ participants, ȱ as ȱ well ȱ as ȱ via ȱ the ȱ weighted ȱ standing ȱ time ȱ (WST) ȱ proxy ȱ for ȱ symptom ȱ severity. ȱ Serum ȱ urea ȱ and ȱ 24 Ȭ hour ȱ urinary ȱ creatinine, ȱ markers ȱ of ȱ nitrogen ȱ metabolism, ȱ were ȱ found ȱ as ȱ the ȱ leading ȱ markers ȱ to ȱ differentiate ȱ ME/CFS ȱ from ȱ health, ȱ as ȱ well ȱ as ȱ degree ȱ of ȱ symptom ȱ severity. ȱ The ȱ role ȱ of ȱ the ȱ cytokine ȱ activin ȱ B ȱ as ȱ a ȱ serum ȱ marker ȱ was ȱ further ȱ examined ȱ along ȱ with ȱ the ȱ range ȱ of ȱ pathology ȱ tests, ȱ and ȱ was ȱ 2 Diagnostics ȱ 2020 , ȱ 10 , ȱ 92 ȱ ȱ found ȱ to ȱ be ȱ significantly ȱ reduced ȱ in ȱ the ȱ serum ȱ of ȱ ME/CFS ȱ patients, ȱ in ȱ addition ȱ to ȱ being ȱ useful ȱ in ȱ differentiating ȱ moderate ȱ to ȱ severe ȱ symptom ȱ severity ȱ when ȱ added ȱ to ȱ RF ȱ models. ȱ The ȱ identification ȱ of ȱ nitrogen ȱ markers ȱ within ȱ pathology ȱ test ȱ results ȱ link ȱ to ȱ deeper ȱ metabolomic ȱ analyses ȱ in ȱ samples ȱ from ȱ ME/CFS ȱ patients, ȱ as ȱ demonstrated ȱ by ȱ previous ȱ results ȱ from ȱ Gooley, ȱ Armstrong, ȱ and ȱ McGregor, ȱ who ȱ reported ȱ abnormalities ȱ in ȱ urea ȱ cycle ȱ metabolites. ȱ Further ȱ work ȱ by ȱ McGregor ȱ et ȱ al. ȱ [9] ȱ is ȱ presented ȱ here, ȱ which ȱ focused ȱ on ȱ biochemical ȱ alterations ȱ during ȱ self Ȭ reported ȱ PEM ȱ episodes. ȱ Glycolytic ȱ anomalies ȱ were ȱ indicated ȱ by ȱ glucose:lactate ȱ ratios, ȱ which ȱ correlated ȱ with ȱ a ȱ fall ȱ in ȱ the ȱ purine ȱ metabolite ȱ hypoxanthine. ȱ A ȱ “hypermetabolic ȱ event” ȱ was ȱ suggested ȱ by ȱ increases ȱ in ȱ the ȱ urinary ȱ excretion ȱ of ȱ methyl Ȭ histidine, ȱ mannitol, ȱ and ȱ acetate. ȱ In ȱ addition ȱ to ȱ these ȱ observations, ȱ data ȱ indicated ȱ a ȱ role ȱ for ȱ hypoacetylation, ȱ showing ȱ that ȱ multiple ȱ biochemical ȱ events ȱ from ȱ histone ȱ function ȱ to ȱ physical ȱ gut ȱ and ȱ muscle ȱ symptoms ȱ coincide ȱ with ȱ PEM. ȱ The ȱ metabolomic ȱ biochemistry ȱ theme ȱ is ȱ further ȱ explored ȱ by ȱ Kashi ȱ et ȱ al. ȱ [10], ȱ who ȱ propose ȱ the ȱ indolamine Ȭ 2,3 Ȭ dioxygenase ȱ (IDO) ȱ metabolic ȱ trap ȱ hypothesis. ȱ The ȱ hypothesis ȱ explores ȱ the ȱ link ȱ of ȱ IDO ȱ biochemistry ȱ in ȱ the ȱ context ȱ of ȱ kynurenine ȱ pathways, ȱ and ȱ the ȱ amino ȱ acid ȱ transporter ȱ LAT1, ȱ through ȱ mathematical ȱ models ȱ of ȱ tryptophan ȱ metabolism. ȱ The ȱ formulation ȱ of ȱ the ȱ IDO ȱ hypothesis ȱ eventuated ȱ from ȱ understanding ȱ the ȱ history ȱ of ȱ outbreaks ȱ world Ȭ wide, ȱ database ȱ searches ȱ for ȱ common ȱ “damaging” ȱ mutations ȱ in ȱ human ȱ enzymes, ȱ and ȱ the ȱ synthesis ȱ of ȱ the ȱ hypothetical ȱ implications ȱ for ȱ ME/CFS ȱ through ȱ mathematical ȱ models. ȱ For ȱ example, ȱ the ȱ balance ȱ of ȱ tryptophan ȱ “steady Ȭ state” ȱ as ȱ physiological ȱ or ȱ pathological ȱ outcomes ȱ is ȱ presented, ȱ with ȱ the ȱ authors ȱ extending ȱ into ȱ experimental ȱ designs ȱ to ȱ test ȱ their ȱ hypothesis. ȱ As ȱ a ȱ disease ȱ with ȱ a ȱ “trigger”, ȱ the ȱ disruption ȱ of ȱ steady Ȭ states, ȱ and ȱ thereafter ȱ perturbations ȱ in ȱ metabolism, ȱ these ȱ characteristics ȱ fit ȱ our ȱ understanding ȱ of ȱ ME/CFS ȱ aetiology. ȱ Concluding ȱ Remarks ȱ To ȱ reiterate, ȱ this ȱ book, ȱ as ȱ a ȱ Special ȱ Issue ȱ of ȱ the ȱ MDPI ȱ journal ȱ Diagnostics , ȱ stands ȱ as ȱ a ȱ landmark ȱ to ȱ consolidate ȱ the ȱ extent ȱ and ȱ value ȱ of ȱ biomedical ȱ research ȱ into ȱ ME/CFS, ȱ and ȱ associated ȱ clinical ȱ observations. ȱ Another ȱ metaphor ȱ may ȱ be ȱ a ȱ “rallying ȱ point”, ȱ especially ȱ for ȱ those ȱ who ȱ have ȱ accepted ȱ the ȱ physiological ȱ basis ȱ of ȱ ME/CFS, ȱ but ȱ have ȱ been ȱ discouraged ȱ by ȱ ongoing ȱ disagreement ȱ among ȱ the ȱ research ȱ community ȱ and ȱ health ȱ professionals. ȱ In ȱ spite ȱ of ȱ debate, ȱ the ȱ evidence ȱ presented ȱ here ȱ and ȱ elsewhere ȱ provides ȱ sufficient ȱ impetus ȱ to ȱ explore ȱ ME/CFS ȱ as ȱ a ȱ biomedical ȱ challenge ȱ that ȱ can ȱ be ȱ solved. ȱ Guided ȱ by ȱ the ȱ journal ȱ title, ȱ all ȱ contributors ȱ were ȱ encouraged ȱ to ȱ focus ȱ on ȱ elements ȱ within ȱ their ȱ research ȱ or ȱ practice ȱ that ȱ emphasised ȱ diagnostic ȱ utility ȱ and ȱ innovation. ȱ Having ȱ succeeded ȱ in ȱ presenting ȱ a ȱ collection ȱ of ȱ manuscripts ȱ spanning ȱ patient ȱ experience ȱ to ȱ pathology, ȱ physiology ȱ to ȱ molecular ȱ and ȱ cellular ȱ biology, ȱ we ȱ hope ȱ that ȱ this ȱ publication ȱ invites ȱ further ȱ insights ȱ from ȱ biomedical ȱ science, ȱ and ȱ finally ȱ acceptance ȱ that ȱ ME/CFS ȱ is ȱ a ȱ true ȱ disease ȱ with ȱ physiological ȱ foundations. ȱ Conflicts ȱ of ȱ Interest: ȱ The ȱ authors ȱ declare ȱ no ȱ conflict ȱ of ȱ interest. ȱ References ȱ 1. Cortes ȱ Rivera, ȱ M.; ȱ Mastronardi, ȱ C.; ȱ Silva Ȭ Aldana, ȱ C.T.; ȱ Arcos Ȭ Burgos, ȱ M.; ȱ Lidbury, ȱ B.A. ȱ Myalgic ȱ Encephalomyelitis/Chronic ȱ Fatigue ȱ Syndrome: ȱ A ȱ Comprehensive ȱ Review. ȱ Diagnostics ȱ 2019, ȱ 9, ȱ 91. ȱ 2. Missailidis, ȱ D.; ȱ Annesley, ȱ S.J.; ȱ Fisher, ȱ P.R. ȱ Pathological ȱ Mechanisms ȱ Underlying ȱ Myalgic ȱ Encephalomyelitis/Chronic ȱ Fatigue ȱ Syndrome. ȱ Diagnostics ȱ 2019 , ȱ 9 , ȱ 80. ȱ 3. Sweetman, ȱ E.; ȱ Noble, ȱ A.; ȱ Edgar, ȱ C.; ȱ Mackay, ȱ A.; ȱ Helliwell, ȱ A.; ȱ Vallings, ȱ R.; ȱ Ryan, ȱ M.; ȱ Tate, ȱ W. ȱ Current ȱ Research ȱ Provides ȱ Insight ȱ into ȱ the ȱ Biological ȱ Basis ȱ and ȱ Diagnostic ȱ Potential ȱ for ȱ Myalgic ȱ Encephalomyelitis/Chronic ȱ Fatigue ȱ Syndrome ȱ (ME/CFS). ȱ Diagnostics ȱ 2019 , ȱ 9 , ȱ 73. ȱ 4. Twisk, ȱ F. ȱ Myalgic ȱ Encephalomyelitis ȱ or ȱ What? ȱ The ȱ International ȱ Consensus ȱ Criteria. ȱ Diagnostics ȱ 2019 , ȱ 9 , ȱ 1 ȱ 5. Vink, ȱ M.; ȱ Vink Ȭ Niese, ȱ F. ȱ Work ȱ Rehabilitation ȱ and ȱ Medical ȱ Retirement ȱ for ȱ Myalgic ȱ Encephalomyelitis/Chronic ȱ Fatigue ȱ Syndrome ȱ Patients. ȱ A ȱ Review ȱ and ȱ Appraisal ȱ of ȱ Diagnostic ȱ Strategies. ȱ Diagnostics ȱ 2019 , ȱ 9 , ȱ 124. ȱ 3 Diagnostics ȱ 2020 , ȱ 10 , ȱ 92 ȱ ȱ 6. Holtzman, ȱ C.S.; ȱ Bhatia, ȱ S.; ȱ Cotler, ȱ J.; ȱ Jason, ȱ L.A. ȱ Assessment ȱ of ȱ Post Ȭ Exertional ȱ Malaise ȱ (PEM) ȱ in ȱ Patients ȱ with ȱ Myalgic ȱ Encephalomyelitis ȱ (ME) ȱ and ȱ Chronic ȱ Fatigue ȱ Syndrome ȱ (CFS): ȱ A ȱ Patient Ȭ Driven ȱ Survey. ȱ Diagnostics ȱ 2019 , ȱ 9 , ȱ 26. ȱ 7. Nacul, ȱ L.; ȱ de ȱ Barros, ȱ B.; ȱ Kingdon, ȱ C.C.; ȱ Cliff, ȱ J.M.; ȱ Clark, ȱ T.G.; ȱ Mudie, ȱ K.; ȱ Dockrell, ȱ H.M.; ȱ Lacerda, ȱ E.M. ȱ Evidence ȱ of ȱ Clinical ȱ Pathology ȱ Abnormalities ȱ in ȱ People ȱ with ȱ Myalgic ȱ Encephalomyelitis/Chronic ȱ Fatigue ȱ Syndrome ȱ (ME/CFS) ȱ from ȱ an ȱ Analytic ȱ Cross Ȭ Sectional ȱ Study. ȱ Diagnostics ȱ 2019 , ȱ 9 , ȱ 41. ȱ 8. Lidbury, ȱ B.A.; ȱ Kita, ȱ B.; ȱ Richardson, ȱ A.M.; ȱ Lewis, ȱ D.P.; ȱ Privitera, ȱ E.; ȱ Hayward, ȱ S.; ȱ de ȱ Kretser, ȱ D.; ȱ Hedger, ȱ M. ȱ Rethinking ȱ ME/CFS ȱ Diagnostic ȱ Reference ȱ Intervals ȱ via ȱ Machine ȱ Learning, ȱ and ȱ the ȱ Utility ȱ of ȱ Activin ȱ B ȱ for ȱ Defining ȱ Symptom ȱ Severity. ȱ Diagnostics ȱ 2019 , ȱ 9 , ȱ 79. ȱ 9. McGregor, ȱ N.R.; ȱ Armstrong, ȱ C.W.; ȱ Lewis, ȱ D.P.; ȱ Gooley, ȱ P.R. ȱ Post Ȭ Exertional ȱ Malaise ȱ Is ȱ Associated ȱ with ȱ Hypermetabolism, ȱ Hypoacetylation ȱ and ȱ Purine ȱ Metabolism ȱ Deregulation ȱ in ȱ ME/CFS ȱ Cases. ȱ Diagnostics ȱ 2019 , ȱ 9 , ȱ 70. ȱ 10. Kashi, ȱ A.A.; ȱ Davis, ȱ R.W.; ȱ Phair, ȱ R.D. ȱ The ȱ IDO ȱ Metabolic ȱ Trap ȱ Hypothesis ȱ for ȱ the ȱ Etiology ȱ of ȱ ME/CFS. ȱ Diagnostics ȱ 2019 , ȱ 9 , ȱ 82. ȱ ȱ © ȱ 2020 ȱ by ȱ the ȱ authors. ȱ Licensee ȱ MDPI, ȱ Basel, ȱ Switzerland. ȱ This ȱ article ȱ is ȱ an ȱ open ȱ access ȱ article ȱ distributed ȱ under ȱ the ȱ terms ȱ and ȱ conditions ȱ of ȱ the ȱ Creative ȱ Commons ȱ Attribution ȱ (CC ȱ BY) ȱ license ȱ (http://creativecommons.org/licenses/by/4.0/). ȱ ȱ 4 diagnostics Review Myalgic Encephalomyelitis / Chronic Fatigue Syndrome: A Comprehensive Review Mateo Cortes Rivera 1, * , Claudio Mastronardi 2 , Claudia T. Silva-Aldana 3 , Mauricio Arcos-Burgos 4 and Brett A. Lidbury 5, * 1 Facultad de Medicina, Grupo de Investigaci ó n Neuros, Universidad del Rosario, Bogot á 110211, Colombia 2 INPAC Research Group, Fundaci ó n Universitaria Sanitas, Bogot á 110211, Colombia 3 Center for Research in Genetics and Genomics-CIGGUR, GENIUROS Research Group, School of Medicine and Health Sciences, Universidad del Rosario, Bogot á 110211, Colombia 4 Group de Investigaci ó n en Psiquiatr í a (GIPSI), Instituto de Investigaciones M é dicas, Facultad de Medicina, Universidad de Antioquia, Medell í n 050002, Colombia 5 The National Centre for Epidemiology and Population Health, RSPH, College of Health and Medicine, The Australian National University, Canberra ACT 2601, Australia * Correspondence: mateo.cortes@urosario.edu.co (M.C.R); brett.lidbury@anu.edu.au (B.A.L.) Received: 22 May 2019; Accepted: 15 July 2019; Published: 7 August 2019 Abstract: Myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS) is a debilitating chronic disease of unknown aetiology that is recognized by the World Health Organization (WHO) and the United States Center for Disease Control and Prevention (US CDC) as a disorder of the brain. The disease predominantly a ff ects adults, with a peak age of onset of between 20 and 45 years with a female to male ratio of 3:1. Although the clinical features of the disease have been well established within diagnostic criteria, the diagnosis of ME / CFS is still of exclusion, meaning that other medical conditions must be ruled out. The pathophysiological mechanisms are unclear but the neuro-immuno-endocrinological pattern of CFS patients gleaned from various studies indicates that these three pillars may be the key point to understand the complexity of the disease. At the moment, there are no specific pharmacological therapies to treat the disease, but several studies’ aims and therapeutic approaches have been described in order to benefit patients’ prognosis, symptomatology relief, and the recovery of pre-existing function. This review presents a pathophysiological approach to understanding the essential concepts of ME / CFS, with an emphasis on the population, clinical, and genetic concepts associated with ME / CFS. Keywords: immunological; chronic fatigue syndrome; myalgic encephalomyelitis; biomarker; neuroimmune; Epstein Barr virus; hypothalamic–pituitary–adrenal axis 1. Introduction Myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS) is a disabling clinical condition characterized by unexplained and persistent post exertional fatigue accompanied by a variety of symptoms related to cognitive, immunological, endocrinological, and autonomous dysfunction [ 1 , 2 ]. The estimated prevalence is estimated at 0.1–0.5% [ 3 , 4 ]. As a result of this debilitating condition, the burden for patients and caregivers is tremendous. In a recent review of the ME / CFS literature reported by The Institute of Medicine (IOM) of the United States (US), it was estimated that between 836,000 and 2.5 million Americans su ff er from ME / CFS, causing an annual financial cost that ranges between 17–24 billion (USD) dollars per year [ 5 ]. The individual income losses are approximately $20,000 annually per household, and the unemployment rates among those who su ff er this pathological condition are between 35–69% [ 5 ]. ME / CFS constitutes a particular enigmatic, debilitating and costly significant public health problem [ 6 ]. It is characterized by a substantial reduction in previous levels Diagnostics 2019 , 9 , 91; doi:10.3390 / diagnostics9030091 www.mdpi.com / journal / diagnostics 5 Diagnostics 2019 , 9 , 91 of occupational, educational, social and personal activities in a patient’s lifetime. The disease a ff ects all ages, races and socioeconomic groups and some studies showed that approximately three to four times as many women as men present the symptoms [ 3 , 7 , 8 ]. Table 1 shows the role of the main tissues involved in the pathogenesis of the disease following the hypothesis of the 3 pillars, explained later in the text. Table 1. Features and function of main tissues representing the three pillars of myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS). HPA: hypothalamic–pituitary–adrenal. System Tissue / Cell Feature Ref. Central Neurological System Neuron The symptomatology is related to a variety of sources of chronic neurological disturbance and associated distortions and chronicity in noxious sensory signaling and neuroimmune activation [9] Glial cells There is a significant blood–brain barrier permeability, microglia activation through toll-like receptors (TLR) signaling, secretion of IL-1B, upregulation of 5-HTT in astrocytes, reduced extracellular 5-HT levels, and hence a reduced activation of 5-HT receptors [10] Immune System Lymphocytes Th1 / Th2 Significant bias toward Th2 immune responses in CFS patients leading to an e ff ector memory cell bias toward type 2 responsiveness [11] NK cells Reduction of cytotoxic activity in CFS, leading to a higher susceptibility of infection [12] B cells Persistence of autoreactive cells that can generate autoantibodies during common infections [13] Endocrine System Hypothalamus– pituitary–adrenal (HPA) axis Enhanced corticosteroid-induced negative feedback, basal hypocortisolism, attenuated diurnal variation, and a reduced responsivity to challenge [14] 2. History In the World Health Organization (WHO) International Classification of Diseases version 2016, both ME and CFS were coded identically and classified as other disorders of the nervous system; nevertheless, “fatigue syndrome”, which non-expert clinicians may view as synonymous with CFS, is classified under mental and behavioral disorders [ 15 ]. This leads to confusion in the classification of the aetiology of the disease, mainly for primary care physicians. Of the two current definitions, myalgic encephalomyelitis (ME) was the first to be defined. In 1934, multiple cases of an unknown illness were recorded around the world. The cases were confused with poliomyelitis or other medullary diseases, but it was eventually di ff erentiated and first known as “neuromyasthenia” [5], with symptom attribution to psychological causes. The details changed from each patient, but in general, patients experienced a variety of symptoms including malaise, tender lymph nodes, sore throat, pain, and signs of encephalomyelitis [ 16 ]. As it occurs presently, the aetiology could not be clearly determined, and it was highly suspected to be infectious because of the flu-like prodrome in most of the a ff ected patients. In 1959, the term “benign myalgic encephalomyelitis” eventually was chosen to reflect an inflammatory disease characterized by severe muscular pains and the evidence of parenchymal damage to the nervous system in the absence of mortality [5]. The landmark case from this era occurred in 1955, and became known as “Royal Free disease” through its association with an English hospital of the same name. Fifty-five nurses, doctors, assistants, and other health personnel were hospitalized on presenting a series of symptoms, which was unusual for the time. Interestingly, most of this hospitalized group contracted upper airway infection prior to the onset of the disease, as well as gastrointestinal alterations, acute vertigo and sore throat, which were followed thereafter by severe headache accentuated by movement and change of position, nuchal pain, pain in the limbs, extreme lassitude, and paranesthesia. Some cases reported more critical symptoms, including muscular cramps and twitching, objective sensory impairments, muscle tenderness, cranial nerve palsies, and ocular movement disorders, suggesting “epidemic neuromyasthenia” [ 17 ]. From that point, the e ff orts to look for the aetiology and the treatment of “myasthenia” began to grow. 6 Diagnostics 2019 , 9 , 91 In the 1970s, the European psychiatric society proposed that myalgic encephalomyelitis (ME) was a psychosocial phenomenon caused by either mass hysteria or the altered medical perception of the community, renaming the disease to “myalgia nervosa”. With this redefinition and no organic explanations of the disease, the medical community began to consider the psychiatric component to understand the condition. This perception among medical professionals vastly limited the research e ff orts to study ME in fields di ff erent from psychiatry and psychology [ 5 , 18 ]. Later on, some researchers demonstrated the severe long-term disability of the disease and abolished the term “benign.” [15]. In 1986, Ramsay [ 17 ] published the first diagnostic criteria for myalgic encephalomyelitis, which is a condition characterized by a unique and chronic form of muscle fatigability even after a minor degree of physical e ff ort, spending three or more days to restore full muscle power. At the end of the 1980s, two cases of an illness resembling mononucleosis attracted the attention of some medical communities [ 19 ]. The disease was then defined as “chronic or recurrent debilitating fatigue and various combinations of other symptoms, including a sore throat, lymph node pain and tenderness, headache, myalgia, and arthralgia” [ 5 ]. Since this time, the illness was largely linked with Epstein-Barr virus (EBV) infection onset and was known as “chronic Epstein-Barr virus syndrome”. In 1987, the US Center for Disease Control and Prevention (CDC) convened a working group to reach a consensus on the clinical criteria of the disease. After much debate about the disease nomenclature, the CDC reached the consensus of “chronic fatigue syndrome”, but noticed that the term “myalgic encephalomyelitis” was the name that was most accepted in other parts of the world. That was the origin of ME / CFS, the term for this condition, that is currently accepted worldwide [ 15 , 20 ]. In 1994 Fukuda proposed a clinical and investigative protocol in order to recreate a comprehensive and integrated approach to study ME / CFS. The definition that was proposed by Fukuda considers chronic fatigue as “self-reported persistent or relapsing fatigue lasting six or more consecutive months” and requires a clinical evaluation to identify or rule out other medical or psychological conditions that may explain the symptomatology [ 21 ]. A diagnosis of ME / CFS involves the absence of other fatigue-associated conditions, unexplained fatigue for at least six months, and at least four of eight minor symptoms. Although this definition had been widely criticized for being overly inclusive, it is still used in the clinical evaluation and diagnosis of the disease. As well as the 1994 criteria, up to 20 other clinical criteria have emerged [ 1 ], with the other notable clinical consensus criteria being the 2003 Canadian Criteria, which was an update in 2011–12 to the International Consensus Criteria [2,22]. ME was recognized by the World Health Organization (WHO) in 1969 as a defined organic neurological disorder. However, in the daily medical practice globally, the concept of ME was not well recognized. The disease formerly described as ‘epidemic neuromyasthenia’ in the US is now more likely to be diagnosed as Chronic Fatigue Syndrome (CFS). Unfortunately, it was not until the ICD-10 that CFS was included in the medical classification of diseases in the US, with inclusions such as benign myalgic encephalomyelitis and post-viral fatigue syndrome [ 23 ]. Also, as a prolonged atypical illness associated with serological evidence of a persistent Epstein-Barr infection, it was associated with infectious mononucleosis syndrome [ 24 ]. However, six years later, Holmes described the first combination of nonspecific symptoms of the syndrome, introducing major and minor criteria for the ME / CFS in the clinical practice [25]. Since the definition of this syndrome, the main issue to diagnose the condition has been the absence of objective parameters to facilitate an accurate clinical assessment of the patient. Patients with ME / CFS were frequently maligned and told they did not have a real physical illness, but rather a psychological condition [ 26 ]. Through years of molecular research and clinical investigation, several clinical definitions have been established in the literature. However, the most widely used in clinical trials have been the Fukuda criteria and the international criteria, both with an inability to separate the ME from the CFS [ 26 , 27 ]. Therefore, a case of ME / CFS is defined by the presence of an unexplained, persistent, and relapsing chronic fatigue of new onset that is not alleviated by rest, which results in a 7 Diagnostics 2019 , 9 , 91 significant reduction in the quality of life, and a concurrent occurrence of four or more of the following eight symptoms that must have persisted during six or more consecutive months: 1. “Brain fog” described as impairment in short-term memory or concentration severe enough to cause a reduction in previous levels of personal activities; 2. A sore throat; 3. Tender cervical or axillary nodes; 4. Muscle pain; 5. Multipoint pain without joint swelling; 6. Headaches; 7. Unrefreshing sleep; 8. Post-exertional malai